The present invention relates to a testing method for sensitivity of anticancer drugs and, in detail, relates to a method in which the sensitivity of cancer cells to anticancer drugs is tested with cancer cells cultured in vitro.
There have been known the following methods for determining the therapeutic efficacy of anticancer drugs, or for the drug-sensitivity test: a clinical testing method in which anticancer drugs are dosed to cancer patients and the change in cancer tissue is examined by diagnostic examination; an in vivo (animal) testing method in which anticancer drugs are dosed to animals to whom human cancer tissue is transplanted; a cell culture testing method in which cancer cells are taken from cancer patients, brought in contact with anticancer drugs and then cultured by using an adequate substrate, or the cells are brought in contact with the drug while cultivating, and after a definite period of time, the growth of cancer cells is measured; and other methods.
It is as usual that after confirmation of the anticancer efficacy of newly developed anticancer drugs by using the cell culture or in vivo-testing methods, the drugs are tested clinically to confirm the efficacy in a final stage, because they can not be applied to the clinical testing method from the first stage of their development. The in vivo-testing method suffers disadvantages of difficulties in controlling animals for experiments such as nude mice etc. and low success rate in the transplantation of human cancer cells, and also requires a long period of time before obtaining experimental results. In contrast, a cell culture testing method has advantages that it is performed by using a relatively simple equipment and by a relatively simple procedure and provides the experimental results in a short period of time. Therefore, an anticancer drug sensitivity test by a cell culture testing method plays a very important role in the development of new anticancer drugs.
Hitherto, as a typical cell culture testing method, a method which is called as the HTCA (Human Tumor Clonogenic Assay) method has been known. According to the method, a single cell suspension is prepared from cancer tissue obtained from a living body and brought in contact with an anticancer drug, and the cancer cells are then cultured in a soft agar substrate. Subsequently, the number of the cancer cell colonies formed after cultivating for a definite period of time is counted to evaluate the inhibition rate on the colony formation by the anticancer drug and thereby the anticancer drug sensitivity is tested. The counting of colonies is performed with eye measurement or by using a colony counter. As alternatives, there are methods in which cells are cultured by using a monolayer culture technique followed by evaluating the efficacy of anticancer drugs against cancer cells using an isotope method and a DNA-measuring method and others.
However, the HTCA method which is the most typical anticancer drug sensitivity testing method in the cell culture methods has the following problems.
First, although the cell culture method using soft agar substrata has an advantage of depressing proliferation of fibroblasts, it requires a large number of cells for culture because of the low colony formation rate of cancer cells. Therefore, the number of tests being carried out with the limited quantity of obtained cells is smaller. Second, there is a problem that the kinds of cancer cells capable of being cultured are limited. That is, the cancer cells variously differ in kind and character due to a tissue difference etc. in a diseased part so that among these variously different kinds of cancer cells only a few kinds of cells can be cultured in a soft agar substrate.
The present inventors, therefore, examined a new anti-cancer drug-sensitivity test, in which a collagen gel substrate was used in place of a soft agar substrate, in a primary culture system using human cancer cells. As a result, we found that even the human cancer cells which did not proliferate in a soft agar substrate were able to proliferate steadily in collagen gel substrate (refer to Koezuka et. al., "Tissue Culture Research Communications", Vol. 6, No. 1, 1987). From the results, the collagen gel culture method made it possible for the cell culture testing method to greatly spread the range of adaptability. Furthermore, the method has been found to have advantages of that the number of cells required for culture is small because cancer cells are proliferated more effectively in collagen gel than in soft agar etc., and that the test results can be obtained effectively within a short period of time.
Generally, when collagen gels are used as substrata, the DNA of cells proliferated in collagen gels is measured for determination of the growth of cancer cells.
However, when cancer cells are cultured in a collagen gel substrate, the collagen gel culture creates a problem that fibroblasts which are contained in cancer tissues also proliferate together with the cancer cells, depending on the kind of the cancer tissue. Since the fibroblasts also possess DNA similarly to the cancer cells, the method for measuring the growth of cancer cells by the above-mentioned quantitative determination of DNA can not be used. Therefore, in such a case the formed cancer cell colonies have to be counted by eye measurement or by means of a colony counter. However, when the measurement is carried out by eye measurement using a stereoscopic microscope, selective counting of the cultured cancer cells alone requires a great deal of labor, and the measurement varies from observer to observer, resulting in inaccurate results. It is also actually impossible to distinguish exactly between the cultured cancer cells and fibroblasts by means of a conventional colony counter. In most human cancer tissues obtained from a surgery material, fibroblasts and cancer cells proliferate simultaneously in collagen gel substrata, and hence it is very difficult to examine the sensitivity of anticancer drugs against human cancer cells cultured in the substrate unless both the fibroblasts and cancer cells are separated. Although several techniques such as the cis-hydroxyproline(Oxyprolin) method, the D-valine method, the citrulline method, and the like have hitherto been proposed as methods for depressing the proliferation of fibroblasts without depressing proliferation of cancer cells, none of these techniques is practically useful because they are effective for only a few, limited kinds of fibroblast.